Capacity affinity sensor

ABSTRACT

This invention describes a capacity affinity sensor based on self-assembled monolayers on an electrode with immobilized recognition elements available to analyte in the surrounding solution. Additional insulation is provided by auxiliary self-assembled molecules. The sensor has exceptional sensitivity and wide operating range due to these parts of the invention. It is versatile because different kinds of recognition elements can be immobilized directly on the surface of the measuring electrode. The electrode then becomes selective to those molecules in the solution, the analytes, that show affinity to the recognition element on the surface. Compared to capacitive sensors described before those described here shows at least a 1000-fold better sensitivity because of the properties of the layer binding the recognition element.

Detecting interactions between molecules forms the basis of manyanalytical methods. The interaction can be detected and quantifiedthrough a number of schemes, e.g. precipitation, separation or throughdifferent marker molecules or reactions. Such an example is thedevelopment of immunoassays during the last three decades, which hasrevolutionized determination of drugs and hormones in clinical andpharmaceutical chemistry as well as contaminants in the environmentalarea. Almost all immunomethods require labels attached either to theantibody or the antigen. Another example is the binding between aDNA-probe and its complementary DNA-strand or DNA-fraction. A number ofreceptors or the complementary molecule can be studied using the sameapproach.

There are a number of disadvantages associated with labels. It they areradioactive the work has to be carried out under strict safety regimeand handling of waste is costly. The use of enzymes as labels requiresan additional time-consuming incubation step. Common for all labels arethat they require a synthetic coupling to either an antigen or anantibody or generally to the recognition element or the analyte. A biglabel may change the affinity between the molecules which is ofparticular concern when an assay is performed by. competition between ananalyte from the sample and an added labeled molecule. Many affinityinteractions cannot be studied because of this. Recognition ofDNA-binding through the use of electrochemical intercalators shows lowsensitivity. Many attempts have therefore been made to detect thebinding itself by potentiometric [Taylor, R. F.; Marenchic, I. G.;Spencer, R. H. Anal. Chim. Acta 1991, 249, 67-70], piezoelectric[Roederer, J. E.; Bastiaans, G. J. Anal. Chem. 1983, 55, 2333-2336], oroptical measurements [Löfås, S. Pure Appl. Chem. 1995, 67, 829-834].

Attempts have previously been made to use capacitance measurements fordetecting molecular interactions without the use of labels. A moleculewith affinity for the analyte should be immobilized on a conductingelectrode surface so that it can interact with the analyte in solutionin such a way that the interaction causes a change in capacitance. Thisprinciple has been used in immunochemistry, by immobilization to oxidesurfaces [Bataillard, P.; Gardies, F.; Jaffrezic-Renault, N.; Martelet,C.; Colin, B.; Mandrand, B. Anal. Chem. 1988, 60, 2374-2379] or forrecognition of DNA-sequences [Souteyrand, E.; Martin, J. R.; Cloarec, J.P.; Lawrence, M. Eurosensors X, The 10th European Conference onSolid-State Transducers, 1996, Leuven, Belgium].

Self-assembled monolayers of thiols, sulfides and disulfides on goldelectrodes have been widely studied and long-chain alkanethiols areknown to form insulating well-organized structures on gold substrates[Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am.Chem. Soc 1987, 109, 3559-3568]. The binding formed between the sulphuratom and gold is very strong and the formed self-assembled monolayers(SAM's) are stable in air, water and organic solvents at roomtemperature [Bain, C. D.; Troughton, E. B.; Tao, Y.-T.; Evall, J.;Whitesides, G. M.; Nuzzo, R. G. J. Am. Chem. Soc. 1989, 111, 321-335].It has been suggested that microcontact printing [Mrksich, M.;Whitesides, G. M. Tibtech 1995, 13, 228-235] and photolithography[Bhatia, S. K.; Hickman, J. J.; Ligler, F. S. J. Am. Chem. Soc. 1992,114, 4432-4433] can be used to pattern surfaces with functionalizedself-assembled monolayers for biosensor production with low cost for adiversity of applications, but until now it has not been possible toproduce direct affinity sensors with high sensitivity.

Terrettaz et al, Langmuir 1993, 9, 1361-1369, discloses a sensor, e.g.for assaying cholera toxin, where the ganglioside GM1 has been bound toa SAM layer. The detection limit for capacitance measurements using thesensor is somewhere within the range from 10⁻⁶ to 10⁻⁹ M. The articlestates that capacitance measurements are unsuitable for assaying choleratoxin because the capacitance changes were too small, and hence, thesensitivity is too low.

Self-assembled monolayers of thiols on gold, with antigenic terminatinggroups have been reported before, but they had coverages of only 14, 19or 31% for different electrodes [Taira, H.; Nakano, K.; Maeda, M.;Takagi, M. Anal. Sci. 1993, 9, 199-206]. The lowest measured value inthe article was at an antibody concentration of 10 ng/ml, which can becompared to 1 pg/ml of antigen measured with our invention (See Example1). The higher sensitivity obtained with our electrode can be explainedby that the gold surface is first covered with a self-assembledmonolayer of a thiol, sulphide or disulphide giving a high coverage ofthe surface, therafter the recognition element is immobilized on thesurface and as the last step the surface is plugged with another thiol.The saturation seems to occur at similar concentrations in the two casesif the larger bulk of the antibody compared to the antigen is taken intoaccount. This comparison thus supports the arguments given above that adense layer is of great importance for a high sensitivity.

DNA-probes have been immobilized e. g to SiO₂ and a sensitivity of 10ng/ml was obtained [Souteyrand, E.; Martin, J. R.; Cloarec, J. P.;Lawrence, M. Eurosensors X, The 10th European Conference on Solid-StateTransducers, 1996, Leuven, Belgium].

A peptide bound to an alkylthiol was also immobilized as aself-assembled layer on gold, but the antibody concentration was in thiscase in the mg/ml range making it a less succesful sensor [Rickert, J.;Wolfgang, G.; Beck, W.; Jung, G.; Heiduschka, P. Biosens. Bioelectron.1996, 11, 757-768].

One of these previous approaches are illustrated in the patent EP244326. The recognition element is bound to an insulating layer on topof a conducting substrate, the insulating layer typically being anoxide. The oxide layer has to be thick, typically 70 nm on silicon, inorder to be stable and sufficiently insulating, resulting in a lowsensitivity. It is difficult to obtain good surface coverage on oxidesand the recognition elements are not well ordered.

Rojas, M.; Königer, R.; Stoddart, F.; Kaifer, A.; J. Am. Chem. Soc.1995, 117, 336-343 discloses an assay method for determining ferrocenein a sample using cyclodextrin. All hydroxy groups of cyclodextin aresubstituted by thiol groups, and the modified cyclodextrins arechemically adsorbed to a gold surface. Empty spaces on the gold surfacebetween the adsorbed modified cyclodextrin molecules are filled withadsorbed pentanethiols. The lowest ferrocene concentration determined is5 μM.

There is always a need for improvements of analysis techniques.Especially when assaying biochemical compounds it is often necessary tobe able to determine concentrations below 1 ng/ml.

SUMMARY OF THE INVENTION

It has now turned out that unexpectedly good capacity affinity sensors,suitable for determining the presence of a certain compound of interestby capacitance measurements using an electrode which can be produced bya method comprising the steps of:

a) providing a piece, of a noble metal where said piece optionally canbe a rod or, alternatively a piece of insulating material such as glass,silica or quartz, on which a noble metal is sputtred or printed;

b) providing a first SAM-forming molecule comprising a coupling groupand/or an affinity group specifically binding said compound of interest;

c) contacting the piece in step a) with the first SAM-forming moleculein step b), thereby obtaining a self-assembling monolayer on said noblemetal surface;

d) in case the first SAM-forming molecule does not comprise an affinitygroup, contacting said self-assembling monolayer on said noble metalpiece with an affinity molecule specifically binding said compound ofinterest, thereby coupling the affinity molecule to the self-assemblingmonolayer; and

e) contacting the piece obtained in step c) or d) with a secondSAM-forming molecule, thereby obtaining a noble metal surface that is atleast 90%, preferably at least 97% covered with a self-assemblingmonolayer.

DETAILED DESCRIPTION OF THE INVENTION

The detection limits reported in this invention are at least threeorders of magnitude better than those reported previously for capacitiveimmunosensors and a comparison is therefore necessary in order toexplain why this invention succeeds so exceptionally well. The insightsbehind this invention are that the recognition layer must be thin,well-ordered and it must cover at least 90%, preferably at least 95%,more preferably at least 97%, and most preferably at least 99% of thesensor surface. In a subsequent step, any free spots between therecognition elements are “plugged”, i.e. covered with a secondself-assembling monolayer-forming molecule, e.g. an alkanethiolcomprising 3-25 carbon atoms preferably in a straight chain, afterobtaining a self-assembling monolayer comprising affinity groups,thereby increasing the tightness and insulation. A capacitive biosensoris covered by an immobilized layer with the recognition element towardthe solution. Electrically it is equivalent to a capacitor between theconducting metal electrode and the conducting solution. Another layerforms when a molecule binds to the recognition element thereby replacingaqueous solution with a non-conducting organic molecule. This isequivalent to the formation of an additional capacitor in series withthe first, thereby decreasing the total capacitance.

Any part of the surface that allows the aqueous solution to penetratebelow the plane where the recognition takes place will act like ashort-circuiting element. The capacitance will therefore increase due tothe higher dielectric constant of the penetrating aqueous solution.Oxide layers are not well ordered and it is therefore impossible to forma dense recognition layer. Self-assembled monolayers are much betterordered and a more perfect coverage can therefore be expected in theimmobilized layers. Furthermore the self-assembled monolayers are muchthinner than the oxide layers, resulting in a larger capacitance inseries with the capacitance formed when molecules bind on the surface.This makes it easier to detect changes in the capacitance when ananalyte binds to the surface.

This invention describes an capacity affinity sensor based onmeasurements of the capacitance change at conducting surfaces. Thegrafted recognition layer should be electrically insulating to preventinterferences from redox couples in the electrolyte solution and highFaradaic background currents. On the other hand, it should be as thin aspossible in order to achieve high sensitivity. The use of self-assembledbinding to gold or other noble metals gives especially thin and compactlayers. The invention also shows how additional insulation can beobtained by plugging with a different type of self-assembling molecule.

Accordingly, the present invention relates to a method for producing acapacity affinity sensor, wherein a piece of a noble metal is coveredwith a layer of a self-assembling monolayer-forming molecule comprisingcoupling groups. Affinity molecules are then coupled to theseself-assembling monolayer-forming molecules. Subsequently any remainingfree spots on the noble metal surface is covered by a secondself-assembling monolayer-forming molecule.

In another aspect, the present invention relates to a capacity affinitysensor comprising a noble metal piece substantially completely coveredwith a self-assembling monolayer comprising first and secondself-assembling monolayer-forming molecules, and where affinitymolecules that specifically binds to a certain molecule of interest havebeen coupled to the first self-assembling monolayer-forming molecules.

In yet another aspect, the present invention relates to a method forqualitatively or quantitatively determining the presence of a certaincompound of interest. A capacity affinity sensor, comprising a noblemetal piece substantially completely covered with a self-assemblingmonolayer comprising first and second self-assembling monolayer-formingmolecules, and where affinity molecules that specifically binds to acertain molecule of interest have been coupled to the firstself-assembling monolayer-forming molecules, is contacted with a liquidsample comprising the compound of interest and the sensor's capacitanceis determined.

In a further aspect, the present invention relates to using said sensorsfor analysing certain compounds of interests, such as human chorionicgonadotropin hormone (HCG), interleukin-2, human serum albumin, atrazineor a certain DNA sequence.

Definitions

As disclosed herein, the terms “self-assembled monolayer” and “SAM” aresynonyms and relates to the spontaneous adsorption of film componentsfrom a solution onto a solid surface making a well-ordered monolayer.Such a layer on gold substrates have previously been describedsubstrates [Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E.D. J. Am. Chem. Soc 1987, 109, 3559-3568].

As disclosed herein, the term “noble metal” relates to a metal chosenfrom the group of gold, silver, copper, platinum and palladium. Gold ispreferred.

As disclosed herein, the term “affinity molecule” relates to a moleculewhich specifically binds to a certain molecule of interest. If themolecule to be determined is an antigen, the affinity molecule might bean antibody, preferably a monoclonal antibody, or an antibody fragmentsuch as a F(ab′)₂ fragment. If a certain nucleic acid sequence is to beidentified, the affinity molecule might be a nucleic acid probespecifically hybridizing to said nucleic acid sequence. The presentinvention can also be used in relation to affinity-mediatingbiomolecules in general, for example in situations where certain nucleicacids bind to antigens other than nucleic acids, such as proteins. Theskilled person is well aware of how to choose suitable affinitymolecules for a certain compound to be determined.

As disclosed herein, the term SAM-forming molecule relates to a moleculehaving the ability of forming a self-assembling monolayer on a noblemetal. A SAM-forming molecule comprises at least one thiol, sulphide ordisulphide group and may optionally also comprise an affinity group.Affinity molecules are coupled to small SAM-forming molecules comprisingcoupling groups in a separate step Examples of such small SAM-formingmolecules comprising coupling groups are thioctic acid and cysteamine.This coupling step is carried out after formation of the self-assemblingmonolayer on the noble metal surface. The skilled person is well awareof how to choose suitable coupling reactions and coupling groups. In thefollowing examples, a self-assembling monolayer consisting of thiocticacid is activated by 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.Subsequently, an affinity molecule is coupled to the activatedmonolayer. However, other similar coupling reactions are described inthe literature.

As disclosed herein, the term “plugging” refers to treatment in asolution containing a thiol, sulphide or disulphide after immobilizationof the affinity molecule to a self-assembling monolayer on a noble metalsurface in order to block any unblocked spots on said surface. Asalready mentioned, it is necessary that the noble metal surface is ascompletely covered by a SAM as possible in order to optimize thesensitivity of the sensor. Suitable examples of SAM-molecules that canbe used for plugging are thiols comprising 3-25 carbon atoms in astraight satured chain. Such SAM-molecules lack coupling groups. Apreferred example is 1-dodecanethiol.

As disclosed herein, SCE stands for the saturated calomel electrode;Potentiostatic perturbation means a fast change in potential; HCG standsfor human chorionic gonadotropin; IL-2 stands for interleukin 2 and HSAstands for human serum albumin.

The interactions that can be measured using this capacitance sensorincludes antigen-antibody, hapten-antibody, peptide-protein, nucleicacids, lectin-hydrocarbon-containing parts, biotin-streptavidin-avidin,receptors-agonist-antagonist, ligand-cells. Complexes can be one part ofthe affinity pair, e. g. hapten-antibody binding to immobilized haptenas recognition element. Fragment, e. g. of antibodies can be usedinstead of the native antibody. Recognition element as used in hereconstitutes any one of the pairs or complexes mentioned above which isimmobilized on the electrode surface. Analyte is the molecule to bedetermined and is normally the other part than the recognition elementin the pairs above.

In this invention a solution containing the molecules, complexes orcells to be determined is allowed to make contact with a surfacecontaining the affinity group, after which the capacitance or impedancechange when an interaction takes place is determined . The capacitancechange takes place between the solution and a metal surface, consistingof solid metal or metal sputtered or printed on an underlayingnon-conducting surface. Faradaic reactions with the metal as well asbackground currents are blocked by the affinity group on the surface,eventually improved by treatment with auxiliary compounds which improvethe insulation. The affinity group is bound to the metal surface, eitherdirectly through self-assembly, or by binding it to a self-assembledcompound on the electrode. It can also be bound through adsorption,polymerization or coating. Measurements are made using electrochemicalperturbations followed by recording of the resulting response. Theperturbations used in the examples described below are potentiostaticsteps or pulses which give rise to current transients from which thecapacitance is evaluated. Perturbations can also be amperometric stepsin which case the change in potential is used for capacitanceevaluation. Perturbations with sinusoidal or other wave-forms have beenreported in the litertature. The sensitivity can be improved by allowinga solution containing a secondary specific ligand to bind to the analytealready on the surface, thereby increasing the size of the boundaggregate and the capacitance change.

The invention will now be described in more detail with reference to theenclosed drawings.

FIG. 1a shows schematically how an antibody can be immobilized to ametal surface. An alkane thiol provides additional insulation. It isalso shown how the total capacitance is made up from a series connectionof those of the double layer, the antibody and the self-assembled layer.

FIG. 1b shows the equivalent circuit used for evaluation of thecapacitance.

FIG. 2 shows the measuring flow cell, a) measuring electrode, b)auxiliary platinum foil electrode, c) platinum wire reference electrode,d) Ag/AgCl reference electrode.

FIG. 3 shows the cyclic voltammetry responses in Fe(CN)₆ ³⁻ when themeasuring electrode was covered with a) thioctic acid, b) thioctic acidand antibody c) thioctic acid, antibody and dodecanethiol. More detailsare given in example 1.

FIG. 4 shows detection of human chorionic gonadotropin hormone (uppercurve) and the the lower curve the absence of response to thenon-specific thyrotropic hormone (lower curve) as specified in example1.

FIG. 5 shows that F(ab′)₂ fragments can be used as recognition elementsfor the human chorionic gonadotropin hormone as described in example 2.

FIG. 6 shows that reduced F(ab′)₂ fragments can be used as recognitionelements for the human chorionic gonadotropin hormone as described inexample 3.

FIG. 7 shows detection of the cytokine Interleukin-2 as mentioned inexample 4.

FIG. 8 shows detection of human serum albumin in a flow cell withdifferent flow rates as discussed in example 5.

FIG. 9 shows the structure of the modified atrazine discussed in example6.

FIG. 10 shows binding of antibodies to atrazines with different sidearms, as discussed in example 6.

FIG. 11 shows the binding of a cytomegalo virus single stranded 179 baseDNA-fragment to an 8 bases long recognition element on the measuringelectrode (upper curve) and the non-specific control with asingle-stranded 207 base DNA fragment from tyrosinase (lower curve). Seeexample 7 for details.

FIG. 12 shows the binding of a cytomegalo virus single stranded 179 baseDNA-fragment to a 25 bases long recognition element on the measuringelectrode. See example 8 for details.

If a solid measuring metal electrode is used, a gold rod typically 3 mmin diameter, is polished, cleaned and coated through self-assembly witha recognition element or with a compound which can be coupled with arecognition element. A great number of coupling methods are known andmay be used as alternatives to those described in the examples. It isalso possible to use metal sputtered or printed on glass, quarts,silicon or another insulating materials as disposable electrodes. Aftercleaning the electrodes are coated in batch and inserted in aquick-connect measuring cell. A number of different recognition elementscan be put on the same sputtered electrode if they are separated byinsulating parts and connected to the potentiostat with switches whichcan be controlled by a microprocessor.

The importance of making the recognition layer thin and with a largecapacitance is illustrated by FIG. 1 with a coupling chemistry as inExample 1. The inverse total capacitance is the sum of the inversecapacitances of each layer in series, i. e. the thioctic acid layer, theantibody layer and the capacitance between the antibody and solution. Ifone of these is small compared to the others, it will dominate the totalcapacitance. Specially if self-assembled parts give rise to a smallcapacitance, it will dominate over the capacitances in the recognitionlayer. Changes in the recognition layer will thus have little effect onthe total capacitance resulting in a low overall sensitivity of thesensor.

The electrode is inserted into a cell which may be either of the cuvettetype or a flow cell as shown in FIG. 2. The cell must contain anauxiliary electrode, typically a platinum foil which should be placedsymmetrically and opposite to the measuring electrode. A referenceelectrode, typically SCE, is placed in the cell so that the voltage dropbetween the reference and measuring electrodes due to capacitive orFaradaic currents becomes very small. In some cases the performance maybe improved if a very small additional reference electrode is used, seeFIG. 1c and the SCE reference is moved away, FIG. 1d. A flow cell givesmore precise control over the mass transfer to the measuring electrodeand injection of sample and cleaning up is more easily automated. Flowcells with volumes of 2 ml and 10 μl were found to have about the samesensitivity. A flow cell with disposable electrodes made by sputteringgold on silicon also had similar properties.

The electrodes are connected to a fast potentiostat which in turn iscontrolled from a microprocessor. The potentiostat will keep themeasuring electrode at a pre-set value versus the reference. Apotentiostatic perturbation is imposed on the measuring electrode. Thecurrents caused by the perturbation voltage are used for evaluation ofthe capacitance of the measuring electrode.

A known volume of sample is normally mixed with a known volume of aconducting liquid in a cuvette in a batch cell. In the case of a flowcell a known volume is injected into a conducting carrier flow pumpedwith a known flow rate. The conducting liquids are normally buffers withionic strengths from a few millimolar and up. The sample can be in anon-conducting medium but a conducting solution must fill the cell whenmeasurements are made.

The invention will now be further described in the following examples.These examples are given for the purpose of illustration and are notintended to limit the scope of the invention.

EXAMPLE 1

The antibody-covered electrode is schematically shown in FIG. 1a. Theelectrode was a gold rod (99.99% Aldrich, 3 mm in diameter) cut up intothin sections threaded to stainless steel holders. Prior toimmobilization the gold rod was polished with alumina slurries down to0.04-0.05 μm. After mounting into the Teflon holder the electrode wasplasma cleaned for 15 min and immediately placed in a solution of 2%(w/w) D/L-thioctic acid in absolute ethanol. The electrode was takenfrom the solution after 24 hours, thoroughly rinsed in absolute ethanoland allowed to dry. Thereafter the electrode was put into a solution of1% (w/w) 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride indried acetonitrile for 5 hours. 5 μl (approximately 1 mg/ml) antibodysolution was placed on the electrode surface and the coupling procedurewas performed at 4° C. for 24 hours. The coupling procedure followedessentially that described by Duan et al [Duan, C.; Meyerhoff, M. E.Anal. Chem. 1994, 66, 1369-1377]. A long thiol, 1-dodecanethiol was usedto “plug”, i.e. block any unblocked spots on the electrode surface.

Capacitance Measurements

The capacitance changes were evaluated from the transient currentresponse obtained when a potentiostatic step was applied to theelectrode. An alternative measurement principle relies on the evaluationof the currents at a number of sinusoidal wave frequencies, usuallycalled impedance spectroscopy. The two methods have been compared usingthe same potentiostat and electrode and found to give almost the sameresults in terms of equivalent capacitances and resistances. Thepotentiostatic step method is faster and more convenient and istherefore used here.

The measuring set-up consisted of a three-electrode system, with anextra reference electrode, connected to a fast potentiostat. Thepotentiostat was connected to a computer (486, 33 MHz) via a Keithley575 measurement and control system, containing 16-bit A/D and D/Aconverters. The Keithley system was powered from the computer through agalvanically isolated power line in the box. The potentiostat waspowered from the Keithley in order to isolate the analog parts from thenoisy digital circuits. The sampling frequency of 50 kHz was determinedby an internal clock in the Keithley box. The current values were takenas the mean of ten repeated steps. The rest potential was 0 mV vs. anAg/AgCl reference electrode. A potential step of 50 mV was applied andthe current transient that followed was sampled. An identical currenttransient but of opposite direction was obtained when the potential wasstepped back to the rest value.

Taking the logarithm of the current gives an almost linear curve fromwhich R_(s) and C₁ can be calculated (see FIG. 1b) using the equation:

i(t)=u/R _(s) exp(−t/R _(s) *C ₁)

where i(t) is the current in the circuit as a function of time; u is theapplied pulse potential; R_(s) is the resistance between the goldsurface and the reference electrode; t is the time elapsed after thepotentiostatic pulse was applied and C₁ is the capacitance measuredbetween the gold electrode and the solution. The first ten currentvalues were used for the calculation and a correlation coefficient ofbetter than 0.99 was obtained.

A platinum wire was used as a reference electrode because it can beplaced closer to the working electrode than a Luggin capillary of glasswithout causing any shielding. This will sharpen the current transientand improve the accuracy of the measurements. The platinum referenceelectrode, though, has no defined potential so its potential wascompared to a commercial Ag/AgCl reference electrode, FIG. 1d, justbefore the potentiostatic pulse was applied.

The carrier solution, 10 mM citrate buffer, pH 7.4 was pumped with aflow rate of approximately 0.5 ml/min through the flow cell. An injectorwith a loop of 250 μl was connected to the flow system.

Cyclic Voltammetry

Cyclic voltammograms were recorded in a three-electrode system in abatch cell. The working electrode was the unmodified or modified goldrod (3 mm in diameter) in a Teflon holder, the auxiliary electrode was aplatinum foil and the reference electrode was a saturated calomelelectrode (SCE). 5 mM of a K₃(Fe(CN)₆) solution was used for themeasurements. The instrumentation used for cyclic voltammetry was aPrinceton Applied 273 A potentiostat controlled by a computer.

A gold surface covered with a long chain alkanethiol layer blocks almostall faradaic currents and is highly insulating with an equivalenttransfer and dynamic resistance of about 2 000 and 69 Ωcm², respectivelyfor a surface covered with butanethiol [Swietlow, A.; Skoog, M.;Johansson, G. Electroanal. 1992, 4, 921-928]. A layer of thioctic acidwas much less insulating with an equivalent transfer and dynamicresistance of 470 and 40 Ωcm², respectively. The permeability of ionsthrough the layer is so high that a redox couple can penetrate it,giving almost the same currents in a cyclic voltammogram as on a baregold electrode, see FIG. 3, curve a. Immobilization of a monoclonalantibody towards human chorionic hormone (HCG) reduces the penetrationof the redox couple, FIG. 3, curve b. Insulation is further improvedwhen the electrode is treated with 1-dodecanethiol as can be seen fromthe absence of redox peaks for such an electrode, FIG. 3, curve c.

Antigen Detection

When an antigen binds to the antibody immobilized on the electrode,there will be an additional layer decreasing the total C₁ further. Thebinding between the antigen and antibody is therefore detected directly.No label is necessary for the antigen. The physical basis for theresponse is thought to arise from displacement of the polar waterfurther out from the electrode surface replacing it with a much lesspolar molecule.

The human chorionic gonadotropin hormone, HCG, was used as modelsubstance. HCG is a glycoprotein with a molecular weight of 30 000 D.The hormone consists of an alpha and a beta chain. The alpha chain isthe same as in the thyrotropic hormone, but the beta chains differ inthe two hormones. The monoclonal antibody immobilized on the electrodewas directed towards the beta chain specific for HCG. Thyrotropichormone and HCG are known to have a cross-reactivity of less than 0.05%[Sigma Chemical Co., Product specification, C-7659]. The thyrotropichormone was used as a control for testing the selectivity of theimmunosensor.

Samples with HCG-concentrations as low as 30 10⁻¹⁵ M (1 pg/ml) wereinjected into the flow system. The capacitance was continuously measuredand found to decrease after an injection until it reached a stablevalue, which took approximately 15 minutes in the 2 ml cell with a flowrate of 0.5 ml/min. The change in capacitance vs. the logarithm of theconcentration, was found to give a linear relationship up to aconcentration of approximately 10⁻¹¹ M (0.3 ng/ml) and to reach asaturating value at 10⁻¹⁰ M, see FIG. 4. The detection limit was around15 10⁻¹⁵ M (0.5 pg/ml) hormone. It was calculated from a comparisonbetween the signal and the irreproducibility of measurements on theantibody surface alone. The irreproducibility corresponds to 15 nFcm⁻².

As usual in flow injection analysis, the sensitivity and detection limitcan be changed by changing the injection volume. A larger sample sizewill thus decrease the detection limit in proportion.

No cross-reactivity whatsoever was observed on the capacity affinitysensor, when the control antigen, thyrotropic hormone, was injected intothe flow system. This suggests that the observed capacitance change isspecific and not caused by an unspecific adsorption of protein to thesensor surface. Injection of a serum sample without added HCG produced a13% increase in the capacitance when the sample entered the cell. Thesignal returned to the previous value when buffer filled the cell again.The increase in capacity is due to the increased ionic strength of thesolution. The experiment thus shows that serum as such does not giverise to any permanent change.

EXAMPLE 2

Capacitance Changes for Antibody Fragments

To increase the sensitivity of the signal, antibodies against HCG weredigested with Ficin to F(ab′)₂ fragments. The idea is to remove aninactive part of the antibody and to move the binding sites closer tothe electrode surface. The fragments were immobilized to the electrodesurface in the same way as described above. The analytical propertieswere similar to those obtained with electrodes covered with the nativeantibody, as shown in FIG. 5. The capacitance, C₁, of the F(ab′)₂electrode was 4500 nFcm⁻² compared to 1400 nFcm⁻² for an electrode witha native antibody. The resistivities were about 63 Ωcm² in both cases.The slopes of the calibration curves were about the same in both casesand an increased sensitivity was not obtained. The increased capacitancewill improve the signal-to-noise ratio somewhat.

EXAMPLE 3

The F(ab′)₂-fragment of HCG was reduced in 0.1 M phosphate buffer, pH 6,containing 0.15 M NaCl, 5 mM EDTA, 4.2 mg/ml 2-mercaptoethylamine during1.5 h at 37° C. The solution was ultrafiltered on an Amicon dialysisfilter, cut-off 10 000 D. The plasma-cleaned gold electrode was dippedinto the filtrate at room temperature over night. The electrode waslater treated with 1-dodecanethiol.

The procedure illustrates a direct binding between the sulfur atom of aunivalent antibody fragment and the metal. The surface will be even morehomogeneous with this procedure and the antibodies' binding part isdirected out into solution. The capacitance will be even higher withthis treatment and the sensitivity will be higher as shown in FIG. 6.

EXAMPLE 4

A monoclonal antibody towards Interleukin-2, IL-2 (M_(w) 15 700 D) wasimmobilized as described above. The results, see FIG. 7, indicate thatthe capacitance change was about half as large for IL-2 as for HCG. Thiscan be explained by the larger molecular weight of HCG.

The IL-2 antibody was taken from a commercial sandwich ELISA kit fordetermination of IL-2 after incubation in micro titer plates with astated detection limit of 6 pg/ml in medium and 10 pg/ml in serum. Thedetection limit for the immunosensor is better than 1 pg/ml. Serumsamples from apparently healthy donors were all below 31 pg/ml [R & DSystems, Inc., Quantikine, IL-2 manual], i.e. the commercial kit couldnot reliably measure IL-2-levels in healthy individuals.

EXAMPLE 5

A monoclonal antibody towards human serum albumin, HSA, (M_(w) 69 000 D)was immobilized on the electrode as described above. The response forHSA was lower than for IL-2, which suggests that more factors thanmolecular size has to be taken into account. Such factors can be thestructure of the antigen, that is if it has a compact or a looseconfiguration, charges of the antigen and the affinity constant for theantibody-antigen complex. One possibility is that albumin is penetratedby the aqueous phase resulting in an increased polarity of the antigenlayer. Another possibility is that the antigen binds in such a way thataqueous solution can penetrate between molecules to some extent. Theremight be sterical hindrance for two large HSA molecules to bind to anantibody with about the same molecular weight as the two.

The capacitance changes obtained for different flow rates were studiedfor the HSA system and the results are shown in FIG. 8. The capacitancechange was found to increase from a flow rate of 0.6 ml/min down to 0.15ml/min. A longer residence time in the cell will allow more HSAmolecules to be transported up to the sensor surface by diffusion andhydrodynamic movements in the solution. An increased sensitivity withdecreasing flow rate is therefore generally expected.

A closer look at the curve shapes for HSA at 0.3 and 0.6 ml/min showthat they differ from those of the other antigens and from that of HSAat 0.15 ml/min. The lower flow rate gives HSA more time to interact withthe antibody and to rearrange itself on the sensor surface. Thesensitivity per molecule seems also to increase when the concentrationdecreases.

EXAMPLE 6

The herbicide atrazine is a small molecule and the capacitance changeswill be small if it binds to an antibody on the measuring electrode. Acompetitive assay can be made by binding a bulky molecule to theherbicide and to allow this labeled antigen to compete with analyteantigens. A displacement assay can also be performed thus dispensingwith the need to use labels. In this assay the antigen was bound tocysteamine self-assembled on gold by coupling to a carboxylic group inthe modified antigen with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimidehydrochloride in dried acetonitrile for 5 hours. Three differentside-chains in the antigen were tested, see FIG. 9. The differentmodified antigens were tested by injecting antibodies, see FIG. 10. Itcan be seen that the t-butyl derivative binds more efficiently to theantibody than the others. It saturates and reaches a constant level atlow antibody concentrations. With an antibody saturated surface,addition of analyte antigen will cause the antibody to be displaced tosome degree, proportional to the concentration, to form a solublecomplex. The capacitance will increase when the amount of antibody onthe surface diminishes. There should be room for the hypervariableregion of the antibody to interact with the bound antigen. If theantigens are packed too denseley they may be interspaced with someinactive compounds.

EXAMPLE 7

DNA can be detected by binding a single-stranded DNA-probe to themeasuring electrode. The gold surface was treated prior toimmobilization as described above. Thereafter it was placed in a thiolsolution of 2% (w/w) of cysteamine in ethanol for 16 hours. Afterreaction the electrode was thoroughly rinsed in ethanol and dried. Thecoupling of the oligonucleotide to the phosphorylated 5′ end wasperformed in an 0.1 M imidazole buffer, pH 6-7, containing 0.15 M1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride, at roomtemperature for 16 hours. After reaction the electrode was rinsed inbuffer and placed in the flow-cell.

An oligonucleotide consisting of 8 bases (SEQ.ID.NO.1) displaying thebase sequence of the cytomegalo virus showed capacitance changes when an179 long single-stranded DNA-fragment was injected and hybridized on thesurface, see FIG. 11. The figure also shows the result when a controlconsisting of a 207-base pair long single-stranded fragment fromtyrosinase mRNA was used as sample. The selectivity is indeed very good.

EXAMPLE 8

An oligonucleotide probe comprising 8 nucleotides might bind, at leastwith some of the bases, to sequences which occur randomly in a mixedbiological sample. Another probe consisting of 20 base-pairs(SEQ.ID.NO.2) was therefore immobilized on the measuring electrode inthe same way as described above. The probe was towards the end of thecytomegalo virus fragment. FIG. 12 shows that a response indeed isobtained.

EXAMPLE 9

There might be some disadvantages with probes directed towards the endof a DNA fragment. It was found, however, that with a probe directedtowards a middle section the capacitance change did indeed occur atfirst but the capacitance returned to the original value after some timein the flow. The probe was therefore immobilized so that it would lieflat on the measuring electrode surface.

34 μl of the oligonucleotide 25-mer (SEQ.ID.NO.3) was incubated on icefor 10 min. with 20 μl M NaHCO₃, pH 9.6, 2 μl 8 mM N-bromosuccinimide inwater, and water to a final volume of 200 μl. Thereafter an electrodepretreated with cysteamine, as described above, was dipped in thesolution and the reaction took place at 50° C. during 1 hour.

3 8 base pairs nucleic acid single linear DNA (genomic) NO NO 1 TTAGGAGA8 20 base pairs nucleic acid single linear DNA (genomic) NO NO 2TAGGGAAGGC TGAGTTCTTG 20 25 base pairs nucleic acid single linear DNA(genomic) NO NO 3 TAGGGAAGGC TGAGTTCTTG GTAAA 25

What is claimed is:
 1. A capacity affinity sensor for determining thepresence of and/or the quantity of a compound of interest in a liquidsample, comprising: a noble metal surface; and a first immobilized andelectrically insulating layer having a first measurable capacitance,wherein the first immobilized and electrically insulating layercomprises a first self-assembled monolayer-forming compound havingcoupled thereto an affinity compound, and a second self-assembledmonolayer-forming compound, the noble metal surface being at least 99%covered with the first immobilized and electrically insulating layer;wherein the affinity compound, upon contact with the liquid sample, iscapable of specifically binding the compound of interest so as to form asecond immobilized and electrically insulating layer having a secondmeasurable capacitance.
 2. The capacity affinity sensor of claim 1wherein the noble metal is gold, silver, copper, platinum or palladium.3. The capacity affinity sensor of claim 1 wherein the noble metalsurface is provided by a piece of the noble metal.
 4. The capacityaffinity sensor of claim 3 wherein the piece of a noble metal is in theshape of a rod.
 5. The capacity affinity sensor of claim 1 wherein thenoble metal surface is provided as a layer of the noble metal coating apiece of an electrically insulating material.
 6. The capacity affinitysensor of claim 5 wherein the electrically insulating material is glassor quartz.
 7. The capacity affinity sensor of claim 1 wherein the firstself-assembled monolayer-forming compound is D/L-thioctic acid,activated with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide.
 8. Thecapacity affinity sensor of claim 1 wherein the second self-assembledmonolayer-forming compound is a thiol comprising 3-25 carbon atoms in astraight saturated chain.
 9. The capacity affinity sensor of claim 8wherein the thiol is 1-dodecanethiol.
 10. The capacity affinity sensorof claim 1 wherein the affinity compound is an antibody, a monoclonalantibody, an antibody fragment or a F(ab′)₂ fragment.
 11. The capacityaffinity sensor of claim 1 wherein the affinity compound is a nucleicacid.
 12. The capacity affinity sensor of claim 1 wherein the affinitycompound is a single-stranded DNA compound.
 13. The capacity affinitysensor of claim 1 wherein the sensor is adapted to determine thepresence of human chorionic gonadotripin hormone (HCG), interleukin-2,human serum albumin, atrazine, or a DNA sequence.
 14. A method forqualitatively or quantitatively determining the presence of a compoundof interest in a liquid sample, comprising the steps of: (a) contactingthe sensor of claim 1 with a reference liquid not containing thecompound of interest and determining the capacitance of the sensor; (b)contacting the sensor with a sample suspected of containing the compoundof interest so as to bind the compound of interest and determining thecapacitance of the sensor having the compound of interest bound thereto;and (c) calculating the difference between the capacitance measured instep (a) and the capacitance measured in step (b) and optionallycalculating the amount of the compound of interest by using prerecordedcalibration data.
 15. The method of claim 14 wherein the compound ofinterest is human chorionic gonadotropin hormone (HCG), interleukin-2,human serum albumin, atrazine, or a DNA sequence.
 16. A capacityaffinity sensor for determining the presence of and/or the quantity of acompound of interest in a liquid sample, comprising: a noble metalsurface; and a first immobilized and electrically insulating layerhaving a first measurable capacitance, wherein the first immobilized andelectrically insulating layer comprises a first self-assembledmonolayer-forming compound and a second self-assembled monolayer-formingcompound, the noble metal surface being at least 99% covered with thefirst immobilized and electrically insulating layer; wherein uponcontact with the liquid sample, the first self-assembledmonolayer-forming compound is capable of specifically binding thecompound of interest so as to form a second immobilized and electricallyinsulating layer having a second measurable capacitance.
 17. A methodfor qualitatively or quantitatively determining the presence of acompound of interest in a liquid sample, comprising the steps of: (a)contacting the sensor of claim 16 with a reference liquid not containingthe compound of interest and determining the capacitance of the sensor;(b) contacting the sensor with a sample suspected of containing thecompound of interest so as to bind the compound of interest anddetermining the capacitance of the sensor having the compound ofinterest bound thereto; and (c) calculating the difference between thecapacitance measured in step (a) and the capacitance measured in step(b) and optionally calculating the amount of the compound of interest byusing prerecorded calibration data.
 18. The method according to claim 17wherein the compound of interest is human chorionic gonadotropin hormone(HCG), interleukin-2, human serum albumin, atrazine, or a DNA sequence.